Characterization of putative circular plasmids in sponge‐associated bacterial communities using a selective multiply‐primed rolling circle amplification

Plasmid transfers among bacterial populations can directly influence the ecological adaptation of these populations and their interactions with host species and environment. In this study, we developed a selective multiply‐primed rolling circle amplification (smRCA) approach to enrich and characterize circular plasmid DNA from sponge microbial symbionts via high‐throughput sequencing (HTS). DNA (plasmid and total community DNA) obtained from sponge (Cinachyrella sp.) samples and a bacterial symbiont (Vibrio sp. CyArs1) isolated from the same sponge species (carrying unknown plasmids) were used to develop and validate our methodology. The smRCA was performed during 16 hr with 141 plasmid‐specific primers covering all known circular plasmid groups. The amplified products were purified and subjected to a reamplification with random hexamer primers (2 hr) and then sequenced using Illumina MiSeq. The developed method resulted in the successful amplification and characterization of the sponge plasmidome and allowed us to detect plasmids associated with the bacterial symbiont Vibrio sp. CyArs1 in the sponge host. In addition to this, a large number of small (<2 kbp) and cryptic plasmids were also amplified in sponge samples. Functional analysis identified proteins involved in the control of plasmid partitioning, maintenance and replication. However, most plasmids contained unknown genes, which could potentially serve as a resource of unknown genetic information and novel replication systems. Overall, our results indicate that the smRCA‐HTS approach developed here was able to selectively enrich and characterize plasmids from bacterial isolates and sponge host microbial communities, including plasmids larger than 20 kbp.     

Do wood-based panels made with agro-industrial residues provide environmentally benign alternatives? An LCA case study of sugarcane bagasse addition to particle board manufacturing

Purpose

Sugarcane bagasse is one of the main agro-industrial residues which can be used to produce wood-based panels. However, more investigations related to its environmental performance assessment are needed, focusing on questions such as: Does it provide environmental benefits? What are its main environmental impacts? Could it substitute wood as raw material? Accordingly, this paper presents a life cycle assessment (LCA) study of particle board manufactured with sugarcane bagasse residues.

Methods

The cradle-to-gate assessment of 1 m³ of particle board made with sugarcane bagasse (PSB) considered three main subsystems: bagasse generation, bagasse distribution, and PSB production. For the inventory of PSB, dataset from two previous LCA studies related to the conventional particle board production and the ethanol life cycle for the Brazilian context were used. The allocation criterion for the bagasse generation subsystem was 9.08 % (economic base). The potential environmental impact phase was assessed by applying the CML and USEtox methods. PSB was compared with the conventional particle board manufactured in Brazil by the categories of the CML and USETox, and including land use indicators. Finally, two scenarios were analyzed to evaluate the influence of the allocation criteria and the consumption of sugarcane bagasse.

Results and discussion

All hotspots identified by CML and USETox methods are mainly related to the PSB production subsystem (24–100 % of impacts) due to heavy fuel oil, electricity, and urea-formaldehyde resin supply chain. The bagasse generation subsystem was more relevant to the eutrophication category (75 % of impacts). The bagasse distribution subsystem was not relevant because the impacts on all categories were lower than 1 %. PSB can substitute the conventional particle board mainly because of its lower contribution to abiotic depletion and ecotoxicity. Regarding land use impacts, PSB showed lower values according to all indicators (38–40 % of all impacts), which is explained by the lower demand for land occupation in comparison to that of the traditional particle board.

Conclusions

PSB can replace the traditional particle board due to its better environmental performance. The analysis of the economic allocation criterion was relevant only for the EP category, being important to reduce diesel and N-based fertilizers use during sugarcane cultivation. Regarding the influence of the sugarcane bagasse consumption, it is suggested that the sugarcane bagasse be mixed up to 75 % during particle board manufacturing so that good quality properties and environmental performance of panels can be provided.     

Methylation-specific PCR: four steps in primer design

Methylation-specific PCR (MSP) is still the method of choice for a single gene methylation study. The proper design of the primer pairs is a prerequisite for obtaining reliable PCR results. Despite numerous protocols describing the rules for MSP primer design, none of them provide a comprehensive approach to the problem. Our aim was to depict a workflow for the primer design that is concise and easy to follow. In order to achieve this goal, adequate tools for promoter sequence retrieval, MSP primer design and subsequent in silico analysis are presented and discussed. Furthermore, a few instructive examples regarding a good versus a poor primer design are provided. Finally, primer design is demonstrated according to the proposed workflow. This article aims to provide researchers, interested in a single gene methylation studies, with useful information regarding successful primer design.     

A sex-associated sequence identified by RAPD screening in gynogenetic individuals of turbot (Scophthalmus maximus)

Understanding the genetic basis of sex determination mechanisms is essential for improving the productivity of farmed aquaculture fish species like turbot (Scophthalmus maximus). In culture conditions turbot males grow slower than females starting from eight months post-hatch, and this differential growth rate is maintained until sexual maturation is reached, being mature females almost twice as big as males of the same age. The goal of this study was to identify sex-specific DNA markers in turbot using comparative random amplified polymorphism DNA (RAPD) profiles in males and females to get new insights of the genetic architecture related to sex determination. In order to do this, we analyzed 540 commercial 10-mer RAPD primers in male and female pools of a gynogenetic family because of its higher inbreeding, which facilitates the detection of associations across the genome. Two sex-linked RAPD markers were identified in the female pool and one in the male pool. After the analysis of the three markers on individual samples of each pool and also in unrelated individuals, only one RAPD showed significant association with females. This marker was isolated, cloned and sequenced, containing two sequences, a microsatellite (SEX01) and a minisatellite (SEX02), which were mapped in the turbot reference map. From this map position, through a comparative mapping approach, we identified Foxl2, a relevant gene related to initial steps of sex differentiation, and Wnt4, a gene related with ovarian development, close to the microsatellite and minisatellite markers, respectively. The position of Foxl2 and Wnt4 was confirmed by linkage mapping in the reference turbot map.     

ABO blood group polymorphisms and risk for ischemic stroke and peripheral arterial disease

Recent studies have demonstrated association between ABO blood system and thrombosis, indicating that individuals belonging to non-O blood groups (A, B or AB) present an increased risk of venous thrombosis, heart disease, and ischemic stroke (IS) as compared to O blood group carriers. In this study, we investigated the frequency of ABO blood group polymorphisms and its association with IS and peripheral arterial disease. Significant differences were observed for O1 (OR 0.57, 95 % CI 0.35–0.95, p < 0.05) and O2 (OR 3.47, 95 % CI 1.15–10.28, p < 0.05) alleles among IS patients while significant differences were observed for B phenotype (26.3 vs 9.5 %, OR 3.42, 95 % CI 1.32–8.76, p = 0.01, patients vs controls, respectively) and alleles A1 (OR 0.31, 95 % CI 0.11–0.84, p < 0.05), O2 (OR 4.61, 95 % CI 1.59–13.23, p < 0.01) and B (OR 3.42, 95 % CI 1.62–7.13, p < 0.001) alleles for PAD patients. O1 allele was an independent variable (OR 0.27, 95 % CI 0.12–0.57, p < 0.001) for IS patients. These data suggest the relationship of non-O blood groups in pathogenesis of thrombosis events and a possible protective effect of O blood group.     

Genetic Identification of Quantitative Trait Loci for Contents of Mineral Nutrients in Rice Grain

In present study, Fe, Zn, Mn, Cu, Ca, Mg, P and K contents of 85 Introgression linee (ILs) derived from a cross between an elite indica cultivar Teqing and the wild rice (Oryza rufipogon) were measured by inductively coupled argon plasma (ICAP) spectrometry. Substantial variation was observed for all traits and most of the mineral elements were significantly positive correlated or independent except for Fe with Cu. A total of 31 putative quantitative trait loci (QTLs) were detected for these eight mineral elements by single point analysis. Wild rice (O. rufipogon) contributed favorable alleles for most of the QTLs (26 QTLs), and chromosomes 1, 9 and 12 exhibited 14 QTLs (45%) for these traits. One major effect of QTL for zinc content accounted for the largest proportion of phenotypic variation (11%-19%) was detected near the simple sequence repeats marker RM152 on chromosome 8. The co-locations of QTLs for some mineral elements observed in this mapping population suggested the relationship was at a molecular level among these traits and could be helpful for simultaneous improvement of these traits in rice grain by marker assisted selection.     

Parasitoid–Pathogen–Pest Interactions of Chelonus insularis, Campoletis sonorensis, and a Nucleopolyhedrovirus in Spodoptera frugiperda Larvae

In this study we examined interactions between two solitary endoparasitoids, the braconid Chelonus insularis and the ichneumonid Campoletis sonorensis, and a multiple-enveloped nucleopolyhedrovirus infecting Spodoptera frugiperda larvae. We examined whether ovipositing females minimize interference by discriminating amongst hosts and examined the outcome of within-host competition between parasitoid species and between the parasitoids and the virus. The egg–larval parasitoid Ch. insularis did not discriminate between virus-contaminated and uncontaminated S. frugiperda eggs; all S. frugiperda larvae that emerged from surface-contaminated eggs died of viral infection prior to parasitoid emergence. The larval parasitoid C. sonorensis also failed to discriminate between healthy and virus-infected S. frugiperda larvae or between larvae unparasitized or parasitized by Ch. insularis. Host larvae parasitized in the egg stage by Ch. insularis were suitable for the development of C. sonorensis when they were multiparasitized by C. sonorensis as first, second, third, and fourth instars, whereas emergence of Ch. insularis was dramatically reduced (by 85 to 100%) in multiparasitized hosts. Nonspecific host mortality was significantly higher in multiparasitized hosts than in singly parasitized hosts. The development time and sex ratio of C. sonorensis in multiparasitized host larvae were unaffected by the presence of Ch. insularis larval stages. Both Ch. insularis parasitized and nonparasitized larvae of the same instar (second, third, or fourth instars) had a similar quantitative response to a challenge of virus inoculum. All host larvae that ingested a lethal dose of virus were unsuitable for Ch. insularis development. In contrast, C. sonorensis did not survive in hosts that ingested a lethal virus dose immediately after parasitism, but parasitoid survival was possible with a 2-day delay between parasitism and viral infection and the percentage of parasitoid emergence increased significantly as the interval between parasitism and viral infection increased. The development time of C. sonorensis was significantly reduced in virus-infected hosts compared to conspecifics that developed in healthy hosts. C. sonorensis females that oviposited in virus-infected hosts did not transmit the virus to healthy hosts that were parasitized subsequently. Field applications of virus for biocontrol of S. frugiperda may lead to substantial mortality of immature parasitoids, although field experiments have not yet demonstrated such an effect.     

Effects of photoperiod on gluconeogenic activity and total lipid concentration in organs of crabs, Neohelice granulata, challenged by salinity changes

Abstract. This study assessed the effects of long (LD) or short (SD) days on the conversion of [¹⁴C]-glycerol to [¹⁴C]-glucose and total lipid concentration in organs of the crab Neohelice granulata challenged by a change in external salinity. In the 20‰-acclimated crabs, no difference was found in the concentration of total lipids in the muscle, hepatopancreas, gills, or hemolymph between crabs acclimated to SD or LD. In SD crabs, the total lipid levels in the anterior and posterior gills did not decrease during an osmotic challenge. Only in the posterior gills did the total lipid levels decrease during acclimation to the 34‰ medium in LD animals. The total lipid concentration in the hemolymph decreased after 1 d of osmotic stress in SD, and increased in the hepatopancreas. In LD crabs, the lipid contents decreased gradually in muscle, and in the hepatopancreas on day 3 after transfer to 34‰ medium. In 20‰-acclimated crabs, the gluconeogenesis activity in both sets of gills was higher in LD than in SD animals. The gluconeogenesis capacity decreased in both sets of gills on the first day of osmotic challenge in SD, and in the posterior gills on the third day in LD crabs. These results suggest that in organs of N. granulata , photoperiod affects the metabolic adjustments to an osmotic challenge.     

Microsatellite loci isolation in the endangered Gran Canarian blue chaffinch (Fringilla teydea polatzeki) and their utility in closely related taxa

The blue chaffinch, Fringilla teydea, is an endemic species of the Canary Islands. This species is formed by two subspecies: The Teneriffean blue chaffinch (F. t. teydea), and the endangered Gran Canarian blue chaffinch, (F. t. polatzeki). Here we report the isolation and characterization of nine tetranucleotide microsatellites (AAAG and AAAT) from the Gran Canarian subspecies, using an enrichment protocol. An average of 7.8 alleles per locus and an average observed heterozygosity of 0.773 were found (n = 28). The loci were tested for their ability to cross amplify in the Teneriffean subspecies and in the common chaffinch (Fringilla coelebs). These microsatellites will be used to manage a captive breeding programme for the endangered Gran Canarian subspecies.